Hsp70 suppresses Vpr-induced cell cycle G₂ arrest. (A) Suppression of Vpr-induced G₂ arrest in fission yeast. Vpr-induced cell elongation was used here as an indication of cell cycle G₂ arrest (11). RE076 is a fission yeast strain that carries a single integrated copy of F34Ivpr (11, 56). RE076+Vector denotes the RE076 strain transformed with the pYZ1N vector control. RE076+spHsp70 and RE076+mHsp70 are RE076 strains transformed with fission yeast or mammalian Hsp70, respectively. vpr-Off, vpr gene expression suppressed; vpr-On, vpr gene expression induced. (B) Hsp70-mediated suppression of Vpr-induced G₂ arrest in 293T-632 cells. Vpr-induced G₂ arrest was measured by flow cytometric analysis after staining DNA with propidium iodide. The extent of G₂ arrest was evaluated by the relative G₂/G₁ ratio (shown in the right-hand panels) between cells cultured in the presence and absence of the inducer (muristerone A): R = [G₂/G₁ (+ inducer)]/[G₂/G₁ (− inducer)] (57). A Western blot (bottom) shows coexpression of HIV-1 vpr and mammalian Hsp70 genes in muristerone A-induced 293T-632 cells. pZY-1 is a vector used for vpr expression, and pZH-1 was used for mHsp70 expression. Both vectors are induced by addition of 1 μM muristerone A (57). Results are shown for one representative experiment out of three performed. (C) Hsp70 suppresses Vpr-induced G₂ arrest in HIV-1-infected cells. MAGI cells were transfected with an Hsp70-expressing vector and 24 h after transfection were infected with a Vpr-positive or Vpr-negative MLV-pseudotyped HIV-1. G₂ arrest was analyzed 48 h after infection by flow cytometry as in panel B. The effect of Hsp70 was evaluated by relative G₂/G₁ ratio (shown in left panels) in cells transfected with Hsp70-expressing vector and control cells: R = [G₂/G₁ (Hsp70−)]/[G₂/G₁ (Hsp70+)]. Results are shown for one representative experiment out of two performed.

Analysis of Hsp70 effects on HIV-1 replication. (A) MAGI cells were transfected with control or Hsp70 siDNA/RNA. Twenty-four hours after transfection, cells were infected with HIV-1 or mock infected. HIV-1 replication was analyzed 48 h after infection by staining intracellular p24 essentially as described previously (32). The percentage of p24-positive cells is shown. For mock-infected cells, only results with Hsp70 siDNA/RNA-transfected cells are presented. (B) The experiment was performed as in panel A, except that cells were infected with Vpr-positive (Vpr+) or Vpr-negative (Vpr−) HIV-1 and extracellular p24 was measured by ELISA. Results show mean ± standard deviation of three independent wells. (C) Triplicate cultures of MAGI cells, transfected with either an Hsp70-expressing (vector+Hsp70) or an empty vector, were infected with Vpr-positive or Vpr-negative HIV-1. Virus replication was measured at indicated times after infection by reverse transcriptase activity in culture supernatants. Results are presented as means ± standard deviation. (D) Triplicate cultures of H9 cells stably transfected with Hsp70-expressing (vector+Hsp70) or an empty vector were infected with Vpr-positive or Vpr-negative HIV-1. p24 in culture supernatants was measured at indicated time points after infection by ELISA. Results are presented as the mean ± standard deviation. The inset in the left panel shows Western blot analysis of Hsp70 and β-actin proteins in transfected H9 cultures.

RNAi-mediated suppression of Hsp70 expression enhances Vpr-specific apoptosis of HIV-1-infected cells. MAGI cells were transfected with Hsp70-specific or control siDNA/RNA duplexes and then infected with Vpr-negative (Vpr−) or Vpr-positive (Vpr+) HIV-1 or mock infected. (A) Hsp70 and Hsp27 were quantified in cell lysates by ELISA 24 h after infection. Results are the mean ± standard error of triplicate wells. (B) Cell cycle distribution was analyzed by FACS 48 h after infection as in Fig. 1B. The effect of siDNA/RNA was evaluated by the ratio of G₂/G₁ in cultures transfected with control siDNA/RNA to G₂/G₁ in cultures transfected with Hsp70-specific siDNA/RNA (shown in the left column panels). Results are representative of two independent experiments. (C) Apoptotic cells were stained with Annexin V-FITC and analyzed by flow cytometry 24 h after infection. The effect of siDNA/RNA was evaluated by the ratio of apoptotic cells in cultures transfected with control and Hsp70-specific siDNA/RNA (shown in left column panels). Results are representative of two independent experiments.

Hsp70 coimmunoprecipitates with Vpr from HIV-1-infected cells. (A) MAGI cells were infected with Vpr-positive (Vpr+) or Vpr-negative (Vpr−) HIV-1. Forty-eight hours after infection, cells were lysed and immunoprecipitated with a polyclonal anti-Hsp70 antibody. The immunoprecipitates were analyzed for the presence of Hsp70 (using anti-Hsp70 MAb) and Vpr (using polyclonal anti-Vpr antibody) by Western blotting (WB). (B). MAGI cells were transfected with Myc-tagged Hsp70 and then infected with Vpr-positive or Vpr-negative HIV-1. Immunoprecipitation was performed with anti-Vpr polyclonal antibody and revealed by anti-Myc MAb.